A. Project Summary/Abstract HIV-1 protease inhibitors (PIs) are the most potent drugs in antiretroviral therapies (ARTs), as protease (PR) is an essential enzyme for HIV. When a PI is used successfully in combination with other anti-HIV drugs, if often can reduce HIV to an undetectable level. However, a successful ART is often threaten by the emergence of viral multidrug resistance to PIs (mdrPRs) that could lead to treatment failure. Consistently, we isolated two mdrPRs (M10PR or M11PR) from HIV-1 infected patients that were resistant to all of the existing PI drugs. The main reason why most of the current PI drugs are unable to inhibit mdrPRs is because these first generation PIs (1stPIs) were designed to ?fit? them into the active enzymatic site by mimicking natural PR substrates. Upon prolonged ART, PR mutations could change shape of the PR active site such that the 1stPIs are no longer able to fit in, thus becoming mdrPRs. To overcome this problem, the second generation PIs (2ndPIs) were designed to ?bind? the active site using P2 ligands based on a new ?backbone-binding? concept by Dr. AK Ghosh. The theory is that if a P2 ligand could firmly binds to its S2 counterpart within the PR active site, it would block the access of the natural viral substrates thus inhibiting the PR activity. Moreover, the tight P2-S2 binding would cling it to the PR active site regardless of its shape changes hence preventing the development of mdrPRs. This new concept led to a FDA-approved drug Darunavir (DRV), which indeed has high genetic barrier to the development of mdrPR and inhibited mdrPRs even when the 1stPIs failed. Unfortunately, DRV was unable to suppress the M10/m11PR either, which underscores the urgent need for new PIs that could combat these mdrPRs. A unique and two-step approach is proposed to meet this need. First, we plan to develop a fission yeast cell-based high-throughput system (HTS) for the new PI discovery. Different from the traditional structure- based drug designs, this method is function-based and has no presumption what kind of PI will be found. Thus, it has the potential to uncover novel PIs. Second, we plan to test some of the new 2ndPIs that were improved beyond DRV, and third generation PIs (3rdPIs) that were with novel designs from Dr. Ghosh's lab. This is a novel approach because no one has ever proposed such a HTS strategy. It is innovative and clinically relevant as it takes advantage of the high HTS efficiency of yeast, uses clinically isolated mdrPRs as the screening targets, and all yeast assays are translatable to mammalian cells. This plan is plausible because we have a very strong and collaborative team to execute the plan, which is built upon our prior success in new PI designs (Dr. AK Ghosh) and fruitful HTS runs conducted at NIH's National Chemical Genomic Center9 (Drs. D. Weber and M. Ferrer). Therefore, we hypothesize that the fission yeast cell-based HTS has the potential to discover novel PIs and to test new PIs with high efficiency. Three Specific Aims are designed to test this hypothesis: 1) adapt the yeast cell-based assays into the HTS formats; 2) conduct HTS for new PIs against the mdrPRs; and 3) characterize the most promising PIs that have the highest therapeutic potentials in combating the mdrPRs.